Human respiratory syncytial virus (HRSV) is the most important viral agent of pediatric respiratory tract disease worldwide and also is important in adults in general and in the elderly and bone marrow transplant recipients in particular. Obstacles to vaccine development include the poor growth of the virus in cell culture, the semi-permissive nature of infection in most animal models, the difficulty of achieving an appropriate balance between immunogenicity and attenuation, and the inefficiency of the immune response in the very young infant. We previously developed a method for producing infectious RSV entirely from cDNA clones (?reverse genetics?), whereby defined changes can be introduced into infectious virus via the cDNA intermediate. We have used this technique extensively to map and characterize RNA signals in the viral genome and to characterize the viral genes and proteins. In addition, reverse genetics is a powerful method for developing live-attenuated vaccine candidates, with the primary focus being the development of an intranasally-administered pediatric vaccine that would be used in conjunction with vaccines for human metapneumovirus and human parainfluenza viruses 1, 2 and 3 that also are under development in the LID/NIAID. One means for attenuating HRSV is based on attenuating point mutations that we identified by sequence analysis of a panel of existing vaccine candidates developed by conventional biological methods. Some of these involve temperature-sensitive (ts) point mutations, each of which is independently attenuating. Ts mutations have the potential for increased growth restriction in the warmer lower respiratory tract and thus might provide increased safety. A second attenuating element is a set of five non-ts point mutations that is attenuating when used together. A third means of attenuation is based on our finding that the NS1, NS2, SH, and M2-2 genes are nonessential and can be deleted individually and in certain combinations to yield viruses that replicate well in vitro but are attenuated in vivo. An advantage of gene deletion mutants is that they should be refractory to reversion. A number of attenuated viruses containing point mutations and/or gene deletions have been constructed. One of these, called rA2cp248/404/1030/delSH, contained four attenuating elements involving point mutations combined with deletion of the SH gene, and was found to have desirable characteristics of attenuation and immunogenicity in infants and young children. However, there was incidence of reversion of a single point mutation involving either of two ts point mutations. This indicated a need for ts point mutations that have increased genetic stability We presently are following a strategy to ?stabilize? these ts amino acid point mutations against loss of the attenuation phenotype. This strategy involves (i) determining the phenotypes associated with every amino acid assignment at the point mutation locus, and (ii) using the degeneracy of the genetic code to choose a codon for an ?attenuating? amino acid assignment that differs by as many nucleotides as possible from all codons for any possible ?non-attenuating? assignments. Thus, the loss of the attenuating assignment would require two or three nucleotide substitutions and will be correspondingly less frequent. Viruses in which the NS1 and/or NS2 genes had been deleted were found to induce the production of large amounts of interferon alpha, beta and lambda in the A549 pnemocyte cell line and in human monocyte-derived macrophages. The NS1 and NS2 proteins were shown to independently and cooperatively interfere with the activation of interferon regulatory factor 3, which is one of the transcription factors involved in inducing the synthesis of interferon alpha and beta. This identified a basis for the attenuation phenotype of these mutations, one that involves alleviating virus-induced interference with the host innate immune response. Because interferons alpha and beta up-regulate both innate and adaptive immunity, HRSV lacking the NS1 and/or NS2 gene has the potential for increased immunogenicity. In contrast, the ts point mutations described above involve the viiral polymerase protein or a transcription gene-start signal, and hence likely exert their attenuating effect by reducing viral RNA synthesis. The non-ts mutations involve the N, F and L proteins, and the basis for their attenuating effect is not known. Yet another basis of attenuation is exemplified by deletion of the M2-2 gene, which alters the viral RNA synthetic program and results in a down-regulation of genome replication and an up-regulation of gene transcription and the synthesis of the viral proteins, including the major protective antigens. An attenuated phenotype that involves increased synthesis of the viral antigens should be particularly advantageous for a live vaccine. Another strategy that has been investigated for attenuating HRSV is to replace one or more genes encoding ?internal? proteins with its counterpart from bovine RSV (BRSV). Examples of replaced genes include the NS1, NS2, N and P genes. The basis for attenuation is the natural host range restriction of BRSV for replication in primates. This showed that substitution of the P gene yielded a promising level of attenuation, whereas substitution of the NS1, NS2 and N genes had small attenuating effects. This provides an additional method for attenuating HRSV based on yet another principle, namely host range restriction.